Metabolic turnover of human c-rasH p21 protein of EJ bladder carcinoma and its normal cellular and viral homologs.

Ulsh, L S; Shih, Thomas Y.

doi:10.1128/mcb.4.8.1647

Cited by 86 publications

(52 citation statements)

References 19 publications

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“…There are only very limited published data on the regulation of stability or production/degradation of the Rho proteins. The phosphorylated form of Ras has been shown to be more stable in cells (26), and carboxyl methylation of RhoA and Cdc42 has been reported to increase their half-lives (39). Our results suggest that phosphorylation of Rho proteins in the C-terminal domain could also protect these proteins from degradation.…”

Section: Pkg Activation Increasesmentioning

confidence: 60%

RhoA Expression Is Controlled by Nitric Oxide through cGMP-dependent Protein Kinase Activation

Sauzeau¹,

Rolli–Derkinderen²,

Marionneau

et al. 2003

Journal of Biological Chemistry

165

128

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Small G proteins of the Rho family function as tightly regulated molecular switches that govern a wide range of cell functions (1). A large body of evidence has now been obtained regarding the important functions of Rho proteins in the vasculature, and RhoA has been shown to play a major role in vascular processes such as smooth muscle cell contraction, proliferation, and differentiation; endothelial permeability; platelet activation; and leukocyte migration (2-4). The activity of Rho is under the direct control of a large set of other regulatory proteins (1). In the inactive GDP-bound form, RhoA is locked in the cytosol by guanine dissociation inhibitors. The guanine nucleotide exchange factors catalyze the exchange of GDP for GTP to activate RhoA (5). Activation is then turned off by GTPase-activating proteins that hydrolyze GTP to GDP. Therefore, both the relative expression of these proteins (in particular, that of RhoA) and the fraction of active GTP-bound RhoA are key determinants of RhoA protein activity.Data are now accumulating regarding the regulation of the amount of active GTP-bound RhoA. In vascular smooth muscle cells, several agonists of G protein-coupled receptors, including thrombin, thromboxane A 2 , endothelin, carbachol, angiotensin, ␣-adrenergic agonists, sphingolipids, and extracellular nucleotides, stimulate RhoA activity through the activation of guanine nucleotide exchange factors and increases in the fraction of GTP-bound RhoA. This RhoA activation is accompanied by the membrane translocation of GTP-bound RhoA (5-8). On the other hand, the NO, cGMP, and cGMP-dependent kinase (PKG) 1 signaling pathway exerts inhibitory action on RhoA functions in cells stimulated by these G protein-coupled receptor agonists. We have previously demonstrated that PKG phosphorylates RhoA at Ser 188 in vitro and that the effects of PKG activation on actin cytoskeleton are lost in cells expressing the non-phosphorylatable RhoA A188 mutant, suggesting that inhibitory effects of PKG on RhoA-mediated contraction and actin organization are due to phosphorylation of RhoA at Ser 188 (9). This effect involves inhibition of membrane translocation of GTP-bound RhoA. Several additional reports have now confirmed the inhibitory effect of the NO/cGMP/PKG signaling pathway on the RhoA-dependent component of agonist-induced contraction (10 -13). Recently, it has also been shown that PKG inhibits RhoA-mediated serum response element (SRE)-dependent transcription (14). PKG inhibits SRE-dependent transcription induced by serum, constitutively active G␣ 12 or G␣ 13 , constitutively active Rho exchange factor p115 RhoGEF , or constitutively active RhoA L63 . This inhibition is associated with a decrease in the amount of active GTP-bound RhoA in cells stimulated with serum or constitutively active G␣ 12 or G␣ 13 , but not in p115 RhoGEF -or RhoA L63 -expressing cells, suggesting that PKG can act both upstream and downstream of RhoA. The effect on steps downstream of RhoA has been confirmed by showing that SRE-dependent transcrip...

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Section: Pkg Activation Increasesmentioning

confidence: 60%

RhoA Expression Is Controlled by Nitric Oxide through cGMP-dependent Protein Kinase Activation

Sauzeau¹,

Rolli–Derkinderen²,

Marionneau

et al. 2003

Journal of Biological Chemistry

165

128

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“…Our data (not shown) indicate that all cytosolic Ras constructs had a half-life comparable to that of membranelocalized H-Ras(Q61L) (i.e. Ϸ22 h (20)) and that our results were therefore unlikely to result from variations in protein stability. We also confirmed that all unprenylated Ras proteins remained cytosolic by cell fixation in formaldehyde followed by immunofluorescent staining as described previously (21), using either anti-HA or anti-pan-Ras antibodies visualized with FITC-conjugated secondary antibody on a Zeiss Axioskop fluorescence microscope.…”

Section: Generation Of Cytosolic Gtp-bound Ras Constructs-mentioning

confidence: 68%

A Distinct Class of Dominant Negative Ras Mutants

Fiordalisi

Holly

Johnson

et al. 2002

Journal of Biological Chemistry

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Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a ␤ 1 -subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Rasinduced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF-or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first extensive characterization of this functionally distinct class of DN Ras inhibitor proteins.The small GTPase, Ras, is involved in numerous aspects of normal cellular metabolism including proliferation, survival/ apoptosis, differentiation, and adhesion/motility (1). Ras must be localized to the inner leaflet of the plasma membrane where, in its GTP-bound active form, it translocates cytosolic effector proteins such as Raf kinase, Ral guanine nucleotide dissociation stimulator (RalGDS), 1 or phosphoinositide 3-kinase (PI3-K) to the membrane for subsequent activation. Ras is anchored at the membrane as the result of a series of posttranslational modifications beginning with the attachment of a farnesyl isoprenoid to a conserved cysteine residue (cysteine 186 in H-and N-Ras; cysteine 185 in K-Ras) located within the "CAAX box," a motif consisting of the carboxyl-terminal four amino acids of Ras. Mutation of the CAAX cysteine therefore prevents both Ras farnesylation and membrane association, leaving the Ras protein unprocessed in the cytosol, where the mutationally ac...

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“…Our results, however, suggest that production of the p21 ras encoded protein as detected by the monoclonal antibody Y13-259 is not restricted to cells which are actively growing or dividing, either in normal mucosa or in tumours where, although identification of clonogenic tumour stem cells is much less easy, the fact that their growth fraction is only in the region of 15% (Wright, 1984) indicates that the pattern of staining observed cannot be ascribed to them alone. In normal crypts the epithelial cell survival time is of the order of 4-8 days (Wright, 1984) whereas the results of in vitro experiments suggest that the half-life of the c-Ha-ras p21 is only 20 h, (Ulsh & Shih, 1984) although this may not be the case in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…GTP dependent phosphorylation of p21 has been shown to be stimulated by both EGF and insulin (Kamata & Feramisco, 1984). The half-life of the cellular p21 in vitro is of the order of 20h (Ulsh & Shih, 1984). Widespread transcription of ras genes has been documented in embryogenesis (Muller et al, 1983) and increased transcription is seen in regenerating liver (Goyette et al, 1984).…”

mentioning

confidence: 99%

Immunocytochemical demonstration of p21 ras family oncogene product in normal mucosa and in premalignant and malignant tumours of the colorectum

Kerr¹,

Lee²,

Quintanilla³

et al. 1985

Br J Cancer

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Metabolic turnover of human c-rasH p21 protein of EJ bladder carcinoma and its normal cellular and viral homologs.

Cited by 86 publications

References 19 publications

RhoA Expression Is Controlled by Nitric Oxide through cGMP-dependent Protein Kinase Activation

RhoA Expression Is Controlled by Nitric Oxide through cGMP-dependent Protein Kinase Activation

A Distinct Class of Dominant Negative Ras Mutants

Immunocytochemical demonstration of p21 ras family oncogene product in normal mucosa and in premalignant and malignant tumours of the colorectum

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